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Protein Kinases Respond to Stress Flashcards

1
Q

What was protein kinases being able to respond to stress identified in?

A

Budding yeast (Saccharomyces Cerevisiae)

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2
Q

How does yeast grow if there is plentiful amounts of glucose?

A

Anaerobically by fermentation (they produce ethanol instead of lactate)

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3
Q

What do yeast do if glucose is absent?

A

They induce genes which encode mitochondrial proteins needed for aerobic metabolism
OR
They switch on genes required for growth on other fermentable carbon sources

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4
Q

Give examples of genes required for growth on other fermentable carbon sources

A

SUC2
MAL
GAL

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5
Q

Function of SUC2 genes

A

Encodes invertase

Needed for the breakdown of sucrose to glucose + fructose

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6
Q

Function of MAL genes

A

Needed for the breakdown of maltose to glucose

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7
Q

Function of GAL genes

A

Needed for the fermentation of galactose

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8
Q

What system do yeast use to detect glucose starvation and switch on the genes required for metabolic changes?

A

SNF1 kinase complex

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9
Q

What is sfn4?

A

A regulator of Snf1 that represses its kinase activity when glucose is present

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10
Q

How was sfn4 function discovered?

A

Overexpression of Snf1 could partly overcome a mutation in Snf4, but not vise versa

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11
Q

What is two-hybrid analysis used for?

A

To test if two proteins interact in vivo

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12
Q

Function of DNA binding domain

A

To bind to DNA at the promoter

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13
Q

Function of activator domain

A

To bind to co-activators to recruit RNA polymerase

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14
Q

How does the two-hybrid analysis work?

A

DNA binding domain is bound to protein A
Activator domain is bound to protein B
If A and B interact, the reporter gene will be switched on, otherwise it will not be

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15
Q

What is Gal4?

A

A protein expressed in yeast

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16
Q

What did the two hybrid analysis reveal in terms of Snf1 and Snf4

A

Snf1 bound to the DNA binding domain of Gal4
Snf4 bound to the activator domain of Gal4
Therefore they work as a complex

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17
Q

Name 3 Snf1-interacting proteins

A

Sip1 (Snf1-interacting protein 1)
Sip2
Gal 83

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18
Q

What is Snf1 and Snf4 unable to grow on?

A

Sucrose, or a non-fermentable carbon source, e.g. ethanol

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19
Q

What happens you delete one of Sip1, Sip2 and Gal83

A

nothing

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20
Q

What happens if you delete Sip1, Sip2 and Gal83 together?

A

You produce the same phenotype as deleting Snf4 or Snf1

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21
Q

What 3 seperate complexes does Snf1 exist as?

A

Snf1:Snf4:Sip1
Snf1:Snf4:Sip2
Snf1:Snf4:Gal83

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22
Q

What are the 3 subunits of AMPK? And what is their ratio?

A

alpha
beta
gamma
1:1:1

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23
Q

What is the alpha subunit of AMPK related to?

A

Snf1

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24
Q

What is the beta subunit of AMPK related to?

A

Sip1/Sip2/Gal83

25
What is the gamma subunit of AMPK related to?
Snf4
26
What isoforms do the AMPK subunits exist as?
alpha1 and alpha 2 beta1 and beta2 gamma1, gamma2 and gamma3
27
How is AMPK activated?
Allosterically by AMP | By upstream kinases which phosphorylate it on Thr172 of the activation loop of the kinase domain
28
Where is Thr172 found?
In Snf1
29
What happens if Thr172 is mutated?
Snf1 function is abolished
30
How many kinases does the yeast genome contain?
120
31
How was Snf1 and AMPK upstream kinase identified?
A library of the 120 yeast strains were made, each overexpression a dingle protein kinase fused to GST All of the 120 GST-kinase fusions were purified on glutathione-sepharose Only Elm1 activated AMPK and Snf1 in cell-free assays KO of Elm1 did not induce a Snf1 phenotype Pak1 and Tos3 (closely related protein kinases) identified KO of all 3 induced a Snf1-like phenotype Adding any back restored growth
32
What was Pak1 renamed to?
Snf1-activating kinase (Sak1)
33
What are the closest relatives of Elm1, Pak1 and Tos3 in the human genome?
Liver kinase B1 (LKB1) | Calmodulin-dependent kinase (CaMKK1/2)
34
What does mutations in LKB1 induce?
``` Peutz-Jeghers Syndrome (inherited susceptibilty to cancer) Lung cancer (20%) Cervical cancer (10%) ```
35
Describe Peutz-Jeghers Syndrome
Patients are heterozygotes Develop frequent benign intestinal tumours and have an increased risk of developing malignant cancers at other sites through loss of their one normal gene copy
36
What 2 accessory subunits are bound to LKB1?
STRAD | MO25
37
What does LBK1:STRAD:MO25 induce?
Phosphorylation and activation of AMPK
38
Name 2 treatments which cause AMPK activation in cells lacking LKB1
AICAR | Phenformin
39
How do treatments induce AMPK activation?
Increase AMP levels
40
When is LKB1 active?
It is always active
41
What does AMP do to AMPK in terms of LKB1?
Makes Thr172 a better substrate for LKB1
42
What does AMP do to AMPK in terms of protein phosphatases?
Makes Thr172 a worse substrate for protein phosphatases
43
Can AMP influence AMPK when it is already phosphorylated?
It can allosterically activate it
44
Why does AMP:ATP increase effect AMPK activation more than ADP:ATP?
Adenylate kinase is activated | This wants to maintain the [ATP][AMP]/[ADP]2 as close to the equilibrium ration of ~1 at all times
45
Is there phosphorylation of AMPK when LKB1 is absent?
Yes, a small amount
46
What increases p-AMPK when LKB1 is absent?
Adding Ca2+ ionophore A23187
47
When is the function of A23187?
Promotes entry of Ca2+ from the extracellular medium, increasing the Ca2+ in the cytoplasm
48
What CaMKK inhibitor inhibited p-AMPK by A23187?
STO-609
49
KO of which CaMKK inhibited AMPK activation by A23187?
CaMKK2
50
What is the second upstream kinase of AMPK?
CaMKK2
51
In cells expressing CaMKK2, what else increases AMPK activation?
Hormones which increase cell Ca2+
52
How do hormones induce intracellular Ca2+?
They bind to receptors which release intracellular Ca2+
53
What does intracellular Ca2+ induce?
A demand for ATP by: - triggering energy-requiring processes such as secretion - the need to remove Ca2+ back out of the cytosol
54
How does AICAR work?
It is a nucleotide which is taken up and converted to AMP
55
How does metformin induce AMPK?
Inhibit mitochondrial ATP synthesis and thus increase AMP
56
What is the issue with metformin?
They deplete ATP and therefore are non-specific and can have off target effects
57
Name a drug which binds to AMPK distally from the AMP binding domain to promote AMPK activation
A-769662
58
Are there any specific AMPK inhibitors and what is used?
No | Compound C is used but this targets other kinases too

Advanced Cell Signalling (12 decks)

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